Proteins are among the most important components of all living systems. Some proteins are hormones; some help defend the body against damage or attack; others act as structural materials of cell walls and membranes, bone and cartilage, hoof and claw. The building blocks of proteins consist of twenty amino acids, linked together by peptide bonds in chains. The diversity in form and function of proteins and peptides stems from the diversity of the amino acid building blocks from which they are made. The twenty naturally-occurring amino acids include side chains that are acidic (Asp and Glu), basic (Lys, Arg, and His), neutral/non-polar (Gly, Ala, Val, Leu, Ile, Phe, Pro, Met), and neutral/polar (Ser, Thr, Tyr, Trp, Mn, Gln, and Cys). The functional nature of a protein is determined by the folded structure that the amino acid polymer assumes. The final three-dimensional form of a protein is largely dependent on its primary structure, i.e. the sequence of the various amino acids along the length of the protein molecule.
Protein Sequencing Technology:
Determining the primary amino acid sequence of any given protein or polypeptide is a formidable task. The first major technology to emerge for the identification of protein sequence is the Edman degradation.1, 2 The Edman degradation method for N-terminal sequence analysis of proteins has been in use for over 50 years. Since the introduction of the spinning cup sequenator,3 automated Edman degradation remains the most widely used method for determining the primary structure of proteins. Extensive research has led to progressively more sensitive Edman sequence analysis. Today's state-of-the-art, gas-phase polypeptide sequencers4 can provide sensitivity at sub-picomole levels. Still, more than 50 years after Edman's initial description of the protocol, the underlying chemistry has remained unchanged: First, a phenylisothiocyanate (PITC) is coupled to the α-amine of the protein or polypeptide to be sequenced. The resulting phenylthiocarbamoyl (PTC) derivative is then hydrolyzed to yield the anilinothiazolinone (ATZ) derivative. The ATZ derivative is then converted in aqueous acid to the more stable phenylthiohydantoin (PTH) (see FIG. 1). In this fashion, the protein or polypeptide target is sequenced, residue-by-residue, starting from the amino terminus of the protein or polypeptide.
In conventional, modern use, the purified protein or polypeptide is applied to glass fiber disks and loaded directly into the reaction chamber cartridge of a gas-liquid solid phase sequencer.4 If the sample is impure, as is commonly the case, gel electrophoresis is usually used to separate the mixture components. Purified protein sample is then transblotted onto chemically inert membranes, which are then placed in the sequencer for analysis.5 
In each degradation cycle, reagents and solvents are delivered to the reaction cell under the control of a microprocessor. Polar reagents are introduced in the gas phase to reduce sample loss. After the cleavage step, the N-terminal ATZ derivative is extracted from the reaction cell and delivered into a conversion flask where it is converted to the more stable PTH amino acid. This final product is subsequently analyzed by HPLC. The elution time of the PTH-amino acid derivative is compared with that of standards to identify each residue.
As delicate as current instruments are, there is still a considerable gap between the demands of protein study and the capabilities available for protein sequencing. First, the condensed-phase Edman degradation process is quite slow. For a gas-phase sequencer, each cycle takes 30-60 minutes to complete. Second, the sensitivity of this technique is insufficient to sequence many important proteins that exist in the cell at sub-femtomole or attomole levels. However, the ability to identify individual components has changed drastically with the recent development of new ionization techniques for mass spectrometry.
Bioanalytical Mass Spectrometry:
Mass spectrometry (MS) is an analytical technique that determines the mass of atoms or molecules by means of ion-field (electric or magnetic) interactions. A mass spectrometer consists of three fundamental components: An ionization source, where gas-phase ions are generated; a mass analyzer, where ions of different mass-to-charge ratios (m/z) are separated; and a detector, where the separated ions produce detectable signals.
Ionization Sources: Over the last two decades, the twin techniques of Matrix-Assisted Laser Desorption/Ionization (MALDI) and Electrospray Ionization (ESI) Mass Spectrometry (MS) were developed.6-8 The two techniques differ significantly but are both highly effective in the production of intact, gas-phase, large biomolecule ions. Producing these ions is a required first step for mass spectrometric analysis.
The success of MALDI is based on the use of a matrix compound that absorbs laser irradiation at a wavelength where the analytes do not. In this technique, the analyte is co-crystallized with a small organic compound. Upon excitation by a laser pulse with sufficient energy density, a sudden and explosive phase transition occurs. From among all the analyte molecules desorbed from the matrix, only a small portion (˜104) are ionized.9 Although the mechanism of ion formation in MALDI remains in debate,10, 11 gas-phase proton transfer is generally believed to be involved in this process. Ions produced in MALDI are usually singly-charged, making MALDI amenable to mixture analysis.
Electrospray ionization results in a distribution of multiply-charged ions for each analyte present. The basic ESI source consists of a metal needle maintained at high voltage (˜4 kV). The needle is positioned in front of a counter-electrode held at ground or low potential (and which also doubles as the inlet of the mass spectrometer). Sample solution is gently pumped through the needle and is transformed into a mist of micrometer-sized droplets that fly rapidly toward the counter electrode (see FIG. 2). In addition to the applied voltage, a concentric flow of nitrogen is often used to help nebulize the solution and dissolve the analyte ions. As each droplet decreases in size, the field density on its surface increases. When charge repulsion exceeds the force of surface tension, the parent droplet splits into smaller daughter droplets. This droplet fission continues until naked ions are formed.
Mass Analyzers: MALDI and ESI have been coupled to many different mass analyzer types. The two most common are the Time Of Flight (TOF) and the Triple Quadrupole (QqQ).
Time-of-flight (TOF) is the simplest mass analyzer, consisting only of a metal flight tube. The mass-to-charge ratios (m/z) of ions are determined by measuring the time it takes the ions to travel from source to detector. In a TOF measurement, an equal amount of kinetic energy is imparted to the analyte ions by placing them in a strong electric field formed by a large DC potential between two plates. Given that all ions of different ink receive the same kinetic energy (qV=mv2/2), low m/z ions will reach the detector sooner than high m/z ions.
Advantages of TOF MS include the capability to deliver complete mass spectra at high speed and with no mass range limit. The mass-resolving power in TOF measurement is, however, limited by the distribution of initial energy in the analyte molecules and the position of the ions prior to acceleration. Typically, the spatial focusing plane in a single-stage mass spectrometer is only a short distance from the acceleration region (i.e., the apparatus has a relatively short focal length), after which the ions will spread out. A two-stage acceleration system is often utilized to allow spatial focusing at a longer distances from the ion source. The spatial focusing plane can be brought to the detector plane by adjustment of the relative field strength between these acceleration stages. Within a certain mass window, energy focusing can be achieved by the technique of delayed extraction, also known as time-lag focusing. The most successful energy focusing method implemented to date is the “reflectron.” In this method, an electrostatic ion mirror (the reflectron) is disposed at the distal end of the flight tube and the electrostatic field within the reflectron is oriented to oppose the acceleration field. Thus, the accelerated ions penetrate into the reflectron, and are ultimately reflected back toward a secondary (or “reflected”) focal point. The more energetic ions penetrate more deeply into the reflectron and hence take longer to be reflected back out of the reflectron. Thus the optics can be adjusted to bring ions of different energies to a space-time focus. While the addition of a mirror provides little improvement in theoretical resolution, it dramatically broadens the mass range of focus.12-14 
A triple quadrupole mass spectrometer is comprised of two mass analyzing quadrupoles (Q1 and Q3) and a radiofrequency-only quadrupole, q2 (see FIG. 3). Quadrupole mass filters can be operated in two basic modes: mass-resolving mode and radio frequency only (RF-only) mode.
In mass-resolving mode, quadrupoles are operated at a constant ratio. The operation points lie on a straight line in a stability diagram, known as the mass scan line (see FIG. 4). When all the experimental parameters are fixed, the mass scan line can be viewed as a collection of points representing particles with different mass-to-charge ratios: heavier ions at the left-lower region and lighter ions at the right-upper region. The portion of the mass scan line that is intercepted by the boundary of the stable region represents a transmission window. Only m/z ratios that fall into this window will be transmitted. The length of this segment defines the resolution of transmission.
In RF-only mode, the DC voltage is removed. The mass scan line in this case coincides with the q axis. The transmission window is now between the m/z of infinity and the low-mass cut-off value. This operation mode is also known as the high-pass mode.
In a QqQ MS, the RF-only quadrupole (q2) functions as a collision cell in which the buffer gas pressure is maintained at about from 1 to about 119 mTorr. Precursor ions selected by Q1 enter the RF collision quadrupole, q2, where they undergo collision-induced dissociation. Product ions are then mass filtered by scanning the third quadrupole, Q3, to produce the product mass spectrum.
Ion Detectors: The most commonly used ion detectors are electron multiplier detectors, including channel electron multipliers (CEM) and microchannel plate detectors (MCP). These detectors operate by means of secondary electron generation. Initial secondary electrons generated upon impact of incident ions start an electron avalanche that produces an output signal. Because the response of electron multiplier detectors to ions with a fixed kinetic energy falls off significantly with increasing mass, ion detectors based on different detection mechanisms have been developed. One strategy is to detect the charge directly. Briefly, as ions approach the detector, image charges are formed on the surface of the detector, which are then picked up by an external circuit generating an output signal. The major limitation in this detection scheme is the low sensitivity due to the lack of inherent amplification.15 In another approach, the energy deposited in a suitable material by impact of an ion can be detected.16-24 Using two superconducting layers separated by an insulating layer, ions that strike the detector create non-thermal phonons (lattice vibrations). Phonons with sufficiently high energy can break the weakly bound electron pairs (Cooper pairs) in the superconducting layer, which results in a measurable tunneling current through the insulating baffler. These detectors are more efficient than MCP's, especially for detecting large ions. However, these types of detectors require liquid helium cooling and generally have a small active area, which limits their use in routine applications.